Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in various biological processes, including fibrinolysis, (Carmeliet et al., 1994, Nature 369:419-424; Carmeliet et al., 1996, Haemostasis 26:132-153; Pinsky et al., 1998, J. Clin. Invest. 102:919-928; Bugge et al., 1996, Proc. Natl. Acad. Sci. USA 93:5899-5904) angiogenesis, (Odekon et al., 1992, J. Cell, Physiol. 150:258-263; Bacharach et al., 1992, Proc. Natl. Acad. Sci. USA 89:10686; Pepper et al., 1990, J. Cell Biol. 111:743-755; Goldberg et al., eds., 1997, In: Regulation of Angiogenesis Birkhauser Verlag, Basel, pp. 391-411) neointima and aneurysm formation, (Clowes et al., 1990, Circ. Res. 67:61-67; Carmeliet et al., 1997, Circ. Res. 81:829-839; Shireman et al., 1997, J. Vasc. Surg. 25:157-164; Noda-Heiny et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:37-43; Lijnen et al., 1998, Arterioscler. Thromb. Vasc. Biol. 18:1035-1045) chemotaxis, (Pedersen et al., 1996, Br. J. Haematol. 95:45-51) and wound healing (Carmeliet et al., 1998, J. Cell Biol. 140:233-245).
Certain of these activities may require the proteolytic activity of uPA, (Kirschheimer et al., 1987, Fed. Am. Soc. of Exper. Biol. and Med. Journal 1:125-128; DePetro et al., 1994, Exp. Cell Res. 213:186-194) whereas others involve intracellular signaling through the urokinase receptor (uPAR) (Pedersen et al., 1996, Br. J. Haematol. 95:45-51; Degryse et al., 1999, Blood 94:649-662) or additional, as yet undefined receptors (Carmeliet et al., 1998, J. Cell Biol. 140:233-245; Kanse et al., 1997, Arteriosclerosis, Thromb., and Vas. Biol. 17:2848-2854; Koopman et al., 1998, J. Biol. Chem. 273:33267-33272; Rabbani et al., 1992, J. Biol. Chem. 267:14151-14156).
Urokinase is synthesized as a single chain molecule (scuPA) which exhibits little or no intrinsic enzymatic activity (Urano et al., 1988, Arch. Biochem. Biophys. 264:222-230; Gurewich et al., 1987, Semin. Thromb. Hemost. 13:146-151; Husain, 1991, Biochemistry 30:5707-5805). scuPA is a multi-domain protein composed of a C-terminal protease domain and an amino-terminal fragment (ATF). The ATF is composed of two domains: a growth factor domain (GFD) which binds to uPAR, and a kringle domain (uPA kringle), the function of which has heretofore been unknown. scuPA can be cleaved by plasmin at the Lys158-Ile159 position to generate an enzymatically active, disulfide-linked two-chain urokinase molecule (tcuPA). Between the ATF and the protease domain is a region designated the connecting peptide (corresponding to amino acids 136-158).
It is hypothesized that the natural enzyme uPA is normally used in the human body to dissolve clots and to facilitate cell migration. Although uPA is known in the art as a useful therapeutic molecule for the treatment of diseases and disorders having as a symptom thereof abnormal clotting in critical blood vessels, there remains a need in the art for compositions and methods which are useful for the treatment of such diseases as well as for the treatment of diseases and disorders having as symptoms abnormally high or abnormally low muscle cell contractility or undesirable angiogenic activity. Such diseases and conditions include the following: cardiovascular diseases and conditions such as hypotension, hypertension and atherosclerosis; thrombotic conditions such as stroke, heart attack and post angioplasty stenting; angiogenic disorders; respiratory diseases and conditions such as pulmonary fibrosis and asthma; diseases and disorders related to tumor cell invasion, angiogenesis and metastasis; wound healing and clotting disorders and reproductive disorders such as premature uterine contraction and impotence. The present invention meets these needs.
The invention includes a composition comprising the urokinase-type plasminogen activator (uPA) kringle in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell or tissue. The uPA kringle shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:1.
In one embodiment, the composition further comprises one or more domains of uPA selected from the group consisting of the growth factor domain, the connecting peptide and the protease domain.
The invention also includes a composition comprising the growth factor domain of uPA in an amount effective to modulate the contractility of a mammalian muscle cell or tissue. The growth factor domain shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:2.
In one embodiment, the composition further comprises one or more domains of uPA selected from the group consisting of the uPA kringle, the connecting peptide and the protease domain.
The invention also includes a composition comprising a polypeptide, the polypeptide (LMW-uPA) comprising the connecting peptide and protease domains of uPA in an amount effective to inhibit the contractility of a mammalian muscle cell or tissue. The polypeptide shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:5.
In one aspect, the cell is in a mammal.
In another aspect, the muscle cell is selected from the group consisting of a smooth muscle cell, a striated muscle cell and a cardiac muscle cell, and the muscle tissue is selected from the group consisting of a smooth muscle tissue, a striated muscle tissue and a cardiac muscle tissue.
In one embodiment, the composition further comprises an inducing compound in an amount effective to mediate the contraction of a mammalian muscle cell or tissue. The inducing compound is selected from the group consisting of phenylepherine, epinepherine, acetylcholine and endothelin.
In another aspect, the composition comprises two chain urokinase (tcuPA). The tcuPA shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:3.
In one embodiment, the composition comprises single chain urokinase (scuPA). The scuPA shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:3.
In another embodiment, the composition comprises the amino terminal fragment (ATF) of uPA. The ATF shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:4.
In one aspect, the uPA kringle is an isolated kringle.
In another aspect, the growth factor domain is an isolated growth factor domain.
In yet another aspect, the ATF is an isolated ATF.
In one embodiment, modulating the contractility of the muscle cell or tissue comprises enhancing or disinhibiting the contractility of the muscle cell or tissue.
In another embodiment, modulating the contractility of the muscle cell or tissue comprises enhancing or disinhibiting the contractility of the muscle cell or tissue.
In one aspect, the cell or tissue is a vascular smooth muscle or endothelial cell or tissue, and the uPA kringle is present in an amount effective to modulate the angiogenic activity of the cell or tissue.
In another aspect, the cell or tissue is a vascular smooth muscle cell or tissue or a vascular endothelial cell or tissue.
In one embodiment, modulating the contractility of the muscle cell or tissue comprises inhibiting the contractility of the muscle cell or tissue.
In another embodiment, modulating the contractility of the muscle cell or tissue comprises inhibiting the contractility of the muscle cell or tissue.
In one aspect, the cell or tissue is a bronchial smooth muscle cell or tissue.
In one embodiment, the composition comprises the deletion mutant polypeptide scuPAxcex94136-143 in an amount effective to enhance or disinhibit the contractility of a mammalian muscle cell or tissue, wherein the scuPAxcex94136-143 shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:6.
In another embodiment, the composition comprises a deletion mutant polypeptide selected from the group consisting of xcex94kringle-scuPA and xcex94kringle-tcuPA in an amount effective to proteolytically activate plasminogen and to inhibit the contractility of a mammalian muscle cell or tissue, wherein the xcex94kringle-scuPA and the xcex94kringle-tcuPA each share at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:7.
In a further embodiment, the composition comprises a polypeptide, the polypeptide comprising the amino terminal fragment (ATF) and the connecting peptide of uPA, wherein the polypeptide shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:8.
In yet a further embodiment, the composition comprises a polypeptide, the polypeptide comprising the uPA kringle and the connecting peptide, wherein the polypeptide shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:9.
In one aspect, the composition is in the form of a pharmaceutical composition.
The invention also includes a composition comprising one or more polypeptides, each of the polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. The one or more polypeptides are present in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell or tissue.
The invention also includes a composition comprising an isolated nucleic acid. The isolated nucleic acid has a nucleotide sequence which shares at least about 75% homology with a nucleotide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. The isolated nucleic acid is present in the composition in an amount effective to transform a mammalian muscle or endothelial cell to provide transgene expression of a polypeptide at a level of expression effective to modulate one or more of the contractility and angiogenic activity of the muscle or endothelial cell after transfection with the isolated nucleic acid.
The invention includes a method of treating a mammal afflicted with a disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal muscle or endothelial cell or tissue angiogenic activity. The method comprises a) administering to the mammal a composition comprising the uPA kringle in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell or tissue, wherein the uPA kringle shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:1; and b) modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue having one or more of abnormal contractility and abnormal angiogenic activity, whereby the disease or condition in the mammal is treated.
In one embodiment, the uPA kringle is a part of a polypeptide which shares at least about 75% homology with a polypeptide selected from the group consisting of SEQ ID NO:3 (tcuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6 (scuPAxcex94136-143), SEQ ID NO:8 and SEQ ID NO:9.
In one aspect, the composition further comprises one or more of an agonist of the uPA kringle, an agonist of a binding protein of the uPA kringle, an antagonist of the uPA growth factor domain, an antagonist of the connecting peptide, an antagonist of a binding protein of the uPA growth factor domain, and an antagonist of a binding protein of the connecting peptide.
In another aspect, the disease or condition is selected from the group consisting of hypotension, hypertension, atherosclerosis, stroke, heart attack, microvascular occlusions, thrombotic microangiopathies, surgically induced thrombotic disorders, angiogenic disorders, pulmonary fibrosis, asthma, tumor cell invasion, tumor cell angiogenesis, tumor cell metastasis, glaucoma diabetic retinopathy, a wound healing or clotting disorder, a uterine contraction disorder and male impotence.
The invention also includes a method for treating a mammal afflicted with a disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal muscle or endothelial cell or tissue angiogenic activity. The method comprises a) administering to the mammal a composition comprising the uPA growth factor domain in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell or tissue, wherein the uPA growth factor domain shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:2; and b) modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue having one or more of abnormal contractility and abnormal angiogenic activity, whereby the disease or condition in the mammal is treated.
In one aspect, the composition comprises the uPA growth factor domain as part of a polypeptide which shares at least about 75% homology with a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:3 (scuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6 (scuPAxcex94136-143), SEQ ID NO:7 (xcex94kringle-scuPA or xcex94kringle-tcuPA) and SEQ ID NO:8.
In one embodiment, the composition further comprises one or more of an agonist of the uPA growth factor domain, an agonist of the connecting peptide, an agonist of a binding protein of the growth factor domain, an agonist of a binding protein of the connecting peptide, an antagonist of the uPA kringle, and an antagonist of a binding protein of the uPA kringle.
In one aspect, the composition is administered to the mammal in an amount effective to inhibit the contractility of a mammalian smooth muscle cell or tissue.
In another aspect, the smooth muscle cell or tissue is a vascular smooth muscle cell or tissue, and the disease or condition treated is hypertension.
In another embodiment, the disease or condition is a respiratory disease or condition selected from the group consisting of asthma, adult respiratory distress syndrome, primary pulmonary hypertension, microvascular thrombotic occlusion and a disorder associated with chronic intrapulmonary fibrin formation.
In one aspect, the uPA kringle is present in an amount effective to inhibit the contractility of a bronchial smooth muscle cell or tissue and is a part of a polypeptide selected from the group consisting of an isolated kringle, ATF, tcuPA, scuPAxcex94136-143, SEQ ID NO:8 and SEQ ID NO:9.
In one embodiment, the disease or condition in the mammal sought to be treated has as a symptom thereof abnormally low vascular smooth muscle cell or tissue contractility.
In another aspect, the uPA kringle is present in an amount effective to enhance or disinhibit the contractility of a vascular smooth muscle cell or tissue and is a part of a polypeptide selected from the group consisting of an isolated kringle, ATF, tcuPA, scuPAxcex94136-143, SEQ ID NO:8 and SEQ ID NO:9.
In one embodiment, the disease or condition has as a symptom thereof abnormally high vascular smooth muscle cell or tissue contractility.
In one aspect, the uPA growth factor domain is present in the composition in an amount effective to inhibit the contractility of a vascular smooth muscle cell or tissue, and is present in the composition as a part of a polypeptide selected from the group consisting of an isolated growth factor domain, scuPA, xcex94kringle-scuPA and xcex94kringle-tcuPA.
The invention also includes a method of identifying a compound which is an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The method comprises a) providing to a first cell and an otherwise identical second cell a composition comprising a polypeptide, the polypeptide comprising one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide, wherein the polypeptide is present in the composition in an amount effective to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue; b) providing to the first cell a test compound; c) assessing the contractility or the angiogenic activity of the first cell and the second cell prior to and after administering the composition and the test compound to the first cell, and prior to and after administering the composition to the second cell; and d) comparing the contractility or angiogenic activity of the first cell with the contractility or angiogenic activity of the second cell prior to and after administration of the composition and the test compound. When the effect of the composition upon contractility or angiogenic activity in the first cell is either increased or decreased relative to the effect of the composition upon contractility or angiogenic activity in the second cell, a compound is identified which is an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue.
The invention also includes a method of treating a disease or condition in a mammal having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The method comprises a) administering to the mammal an amount suspected to be effective for modulating the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue of an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof; b) providing the agonist or antagonist to a muscle or endothelial cell or tissue in the mammal having abnormal contractility or abnormal angiogenic activity, or to a tissue or fluid which is contiguous therewith; and c) modulating the effect of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the muscle or endothelial cell or tissue having abnormal contractility or abnormal angiogenic activity, whereby a disease or condition in the mammal having abnormal muscle cell or tissue contractility or abnormal angiogenic activity as a symptom thereof is treated.
In one embodiment, the disease or condition treated is the vascular disease hypertension.
In another embodiment, the agonist or antagonist is one or more of an antagonist to the uPA kringle, an antagonist to a binding protein of the uPA kringle, an agonist of the uPA growth factor domain, an agonist of a binding protein of the uPA growth factor domain, an agonist of the connecting peptide, and an agonist of a binding protein of the connecting peptide.
In one aspect, the disease or condition treated is selected from the group consisting of asthma, adult respiratory distress syndrome, primary pulmonary hypertension, microvascular thrombotic occlusion and a disorder associated with chronic intrapulmonary fibrin formation.
In another aspect, the agonist or antagonist is one or more of an agonist to the uPA kringle and an agonist to a binding protein of the uPA kringle.
The invention also includes a method of identifying whether a test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide. The method comprises a) assessing the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide upon a first cell or tissue, wherein the first cell or tissue comprises the test protein or is contiguous with a tissue or fluid of a mammal which comprises the test protein; b) assessing the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide upon a second, otherwise identical cell or tissue which does not comprise the test protein and which is not contiguous with a tissue or fluid which comprises the test protein; and c) comparing the contractility modulating effect or the angiogenic activity modulating effect in the first cell or tissue with the contractility modulating effect or the angiogenic activity modulating effect in the second cell or tissue. If the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide is greater in the first cell or tissue relative to the second cell or tissue, then the test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide.
Also included in the invention is a method of identifying a functional element of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide, the functional element participating in the modulation of contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The method comprises a) preparing one or more mutant polypeptides which lack a portion of the amino acid sequence of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide; b) assessing the ability of each of the mutant polypeptides to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue once provided to the cell or tissue, or to a tissue or fluid which is contiguous with the cell or tissue; c) identifying, from b) a mutant polypeptide which is not able to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue; and d) determining from c) and a) the corresponding deleted portion of the amino acid sequence of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide which participates in the modulation of muscle or endothelial cell or tissue contractility or angiogenic activity, whereby a functional element of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide is identified.
The invention also includes a method of treating a vascular disease or condition in a mammal having as a symptom thereof abnormally high fibrin clot formation. The method comprises a) administering to the mammal a composition comprising one or more of xcex94kringle-scuPA, xcex94kringle-tcuPA, an antagonist of the uPA kringle and an antagonist of a binding protein of the uPA kringle in an amount effective to inhibit the contractility of a mammalian vascular smooth muscle cell or tissue, wherein the xcex94kringle-scuPA and xcex94kringle-tcuPA share at least about 75% homology with the polypeptide corresponding to SEQ ID NO:7; b) providing the composition to an affected vascular smooth muscle cell or tissue of the cardiovascular system of the mammal which has or is prone to excessive fibrin clot formation, or to a tissue or fluid which is contiguous therewith; and c) vasodilating the affected vascular smooth muscle cell or tissue by inhibiting the contractility of the affected vascular smooth muscle cell or tissue, thereby promoting both fibrin clot lysis and vasodilation in the affected area of the vasculature of the mammal, thereby treating the vascular disease or condition.
The invention also includes a kit for treating a disease or condition in a mammal, the disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The kit comprises a) a composition comprising a polypeptide, the polypeptide comprising one or more of the uPA kringle, the uPA growth factor domain, and the connecting peptide in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell or tissue; and b) an instructional material.
In one aspect, the kit further comprises a sterile solvent suitable for dissolving or suspending the composition prior to administering the composition to the mammal.